Preparation of porous silicone rubber for growing cells or living tissue

ABSTRACT

A method of making a silicone rubber having a structure adapted for growth of cells or living tissue, which comprises contacting a silicone rubber precursor with a biologically-acceptable sacrificial filler, curing the resultant mixture and removing the sacrificial filler to form a structured silicone rubber. The sacrificial filler is preferably an inorganic salt that has been ground, and the salt is selected from metal halides, metal carbonates and metal bicarbonates.

BACKGROUND OF THE INVENTION

The present invention relates to methods for manufacturing siliconerubber that is adapted to promote cell adhesion and growth, and, inparticular, to methods for providing silicone rubber with a modifiedsurface or structure for enhanced cell attachment. The resultantsilicone rubber is well-suited to a variety of tissue culture andmedical applications.

Silicones surpass other elastomers in many performance categoriesbecause of their rigid silicon-oxygen chemical structure. The process ofvulcanisation transforms this structure, allowing the silicon-oxygenpolymer to become an elastic rubber. Silicone rubbers are stablethroughout a temperature range of −46° C. to 232° C. They are odourless,tasteless and do not support bacterial growth. Silicone rubbers also donot stain or corrode with other materials. Most importantly, siliconerubbers are not physically or chemically degraded or altered by contactwith body fluids, are not toxic or allergenic to human tissue and willnot excite an inflammatory or foreign body reaction. Silicone rubberscan be formulated and tested for full bio-compatibility and compliancewith guidelines for medical products. A further and particularlyimportant advantage of silicone rubbers is that they have the highestoxygen permeability of known polymers.

Forming textured and porous silicone rubber allows all of theseadvantageous properties of silicone rubber to be exploited and enhanced.For example, a textured surface will not only greatly increase theavailable surface area for cell attachment, but will also encourage cellattachment. Furthermore, the increased surface area will increase theoxygen permeating through the silicone, enhancing the metabolic activityof the cells attached thereto. These advantages are very important inthe various applications of textured and porous silicone rubbersdiscussed below.

SUMMARY OF THE INVENTION

In a first aspect of the invention, there is provided a method of makinga silicone rubber having a structure adapted for growth of cells orliving tissue, which comprises contacting a silicone rubber precursorwith a biologically-acceptable sacrificial filler, curing the resultantmixture and removing the sacrificial filler to form a structuredsilicone rubber. Any suitable silicone rubber precursor may be used,depending upon the intended application of the resultant structuredsilicone rubber. Such silicone rubber precursors are widely availablecommercially, for example, from Dow Chemical Corporation, Midland,Mich., USA, or from GE Silicones Europe, Bergen op Zoom, theNetherlands. In a preferred embodiment, the silicone rubber precursor isone that can be cured or vulcanised at room temperature. This obviatesthe need to expose the mixture to elevated temperatures, which isparticularly useful as some sacrificial fillers become unstable anddecompose at elevated temperatures, thus making it difficult to controlthe final form of the structured silicone rubber. In a furtherembodiment, the biologically-acceptable sacrificial filler isbiocompatible, such that it is innately non-toxic and does not leave atoxic residue. This is of particular importance where the structuredsilicone rubber is intended for use in tissue culture and medicalapplications, although a number of further factors also need to beconsidered when choosing a suitable sacrificial filler. For example, thesacrificial filler should preferably not react with the silicone rubber,either in its precursor form or in its cured state. The filler shouldalso preferably be soluble in order to facilitate its removal bydissolution and the solvent used to dissolve the material shouldpreferably not react with the silicone rubber. If the silicone rubber isto be cured at elevated temperatures, it is usually desirable to use asacrificial filler that is stable at the curing temperatures, sincematerials that melt or decompose at high temperatures may be unsuitable,particularly if a structured silicone rubber having a high degree ofregularity is desired. Finally, for commercial reasons, it is generallydesirable that the sacrificial filler should be relatively inexpensiveand readily available. In a preferred embodiment, the sacrificial filleris ground, prior to contacting the silicone rubber precursor. This hasthe advantage of allowing the resultant structure of the silicone rubberto be controlled much more accurately. Any suitable method for grindingthe sacrificial filler may be used, although it has been found thatwet-milling the sacrificial filler, prior to mixing with the siliconerubber precursor, gives good results. However, the sacrificial fillermay also be ground by dry milling, preferably under an inert or dryatmosphere, such as under dry nitrogen or argon gas. In a preferredembodiment, the sacrificial filler is milled to a particle size of0.01-10 μm, preferably 0.05-1 μm, and most preferably 0.1-0.4 μm. In afurther embodiment, the sacrificial filler is granular and, preferably,crystalline, although certain amorphous fillers may also be suitable.Inorganic salts have been found to give particularly good results,although certain crystalline organic compounds, such as simplesaccharides, may often be equally effective. Where the sacrificialfiller is an inorganic salt, it is especially preferred to grind itfirst by milling it in an organic solvent, since this gives good controlover resultant particle size. Preferably, the sacrificial filler is aninorganic salt selected from the group consisting of metal halides,metal carbonates and metal bicarbonates, especially one selected fromthe group consisting of lithium bicarbonate, sodium bicarbonate,potassium bicarbonate, lithium chloride, sodium chloride and potassiumchloride. In an especially preferred embodiment, the sacrificial filleris sodium bicarbonate or sodium chloride, preferably of high purity,such as food grade sodium bicarbonate or sodium chloride. In this lastembodiment, the sodium bicarbonate or sodium chloride is preferablywet-milled under xylene, although other volatile organic solvents mayalso be used. In a further embodiment, the ground sacrificial filler isclassified, prior to contacting the silicone rubber precursor to ensureuniform particle size distribution, for example, by passing the groundmaterial through sieves or by using a Malvern® Particle Sizer. Inanother embodiment, the sacrificial filler is removed by dissolution,preferably in an aqueous solvent. In the latter case, the sacrificialfiller is desirably chosen so that it does not cause swelling of thesilicone rubber when removed with an aqueous solvent. In a furtherembodiment, at least a portion of the free —OH groups that are normallypresent in the silicone rubber are chemically modified, so as to enhanceor promote cell adherence. For example, free —OH groups may bechemically converted to form positively charged groups, for example, byreaction with diethylaminoethylbromide to give DEAE moieties, or to formnegatively charged groups, for example, by reaction with iodoaceticacid, to give carboxylate moieties. In an alternative embodiment, thesurface of the silicone rubber may be charged electrostatically, forexample, by bombardment with electrons. Alternatively, the surfacecharacteristics of the silicone rubber may be modified by applying athin coating of a suitable polymer, so as to make it more adherent tocertain cells, whilst still retaining a sufficient degree of gaspermeability. Any suitable polymer may be used, such as one selectedfrom the group consisting of polyolefins, polyvinyl resins, polyesterresins, polyurethanes, polyamines, polyamides, polyethers andpolysaccharides. In a preferred embodiment, the silicone rubberprecursor also includes at least one additive that is not removed withthe sacrificial filler and serves to impart desired physical propertieson the resultant silicone rubber. For example, the additive may be ametal powder or carbon black, which can be used to render the siliconerubber electrically conductive. Alternatively, the additive may bestainless steel powder or iron oxide, which can be used to increase thedensity of the silicone rubber. The additive may also be an inertsubstance, such as glass, which can be used to render the siliconerubber mechanically rigid. However, many other suitable additives willalso be apparent to those skilled in the art.

In a second aspect of the invention, there is provided a method ofmaking a silicone rubber having a structure adapted for growth of cellsor living tissue substantially in accordance with the invention in itsfirst aspect, wherein a surface of the silicone rubber precursor iscontacted with the sacrificial filler, so as to form a structuredsilicone rubber having a textured surface. The textured surface of thesilicone rubber helps to facilitate attachment of adherent cells, aswell as providing an increased surface area and, thus, number of sitesfor attachment of cells relative to an untextured surface. In anembodiment, the inventive method comprises forming a coating of asilicone rubber precursor on a substrate, contacting a surface of thecoating with a biologically-acceptable sacrificial filler, curing theresultant mixture and removing the sacrificial filler to form a texturedsilicone rubber. Suitable silicone rubber precursors and biologicallyacceptable sacrificial fillers are essentially as described above inrelation to the invention in its first aspect. In a preferredembodiment, the surface of the coating is contacted with the sacrificialfiller under pressure, such that the sacrificial filler is substantiallycompletely embedded in the coating. For example, the sacrificial fillermay be dry-sprayed on to the surface of the coating, or may be appliedloosely to the surface of the coating and then embedded by contactingthe surface with a pressure roller. Preferably, the sacrificial filleris embedded to a depth of 0.1-1.0 mm, more preferably 0.1-0.5 mm, andmore preferably 0.1-0.25 mm. In an alternative embodiment, thesacrificial filler is scattered or sprinkled over the surface of thecoating, such that the sacrificial filler is only partially embedded inthe surface. The latter technique can be used to provide the surface ofthe silicone rubber with a less uniform texture that is particularlysuitable for growing certain types of adherent cells. Preferably, theresultant textured surface is micro-cupulated, i.e., cratered or pitted,the micro-cupules having a depth of less than 1 mm, preferably a depthof 0.5-0.1 mm. In a preferred embodiment, the micro-cupules measure lessthan 2 mm across, preferably less than 1 mm across, and, mostpreferably, less than 0.5 mm across. Silicone rubbers are available witha wide range of different physical properties, both in the uncured andcured state, and their methods of cure also differ widely. Consequently,the nature and properties of the silicone rubber used can affect themanufacturing process and the choice of a suitable silicone rubberprecursor can be important. The silicone rubber precursor should beselected with due consideration to the manner in which the mixture is tobe applied to the substrate, the conditions required for curing, and thedesired properties of the end product. The uncured silicone rubbershould usually have an appropriate viscosity for the method of itsapplication to the substrate, and should retain its general form oncethe sacrificial filler has adhered to its surface. The conditions forcuring must generally be compatible with both the substrate to which theuncured silicone rubber is applied and the sacrificial filler thatadheres to the surface. Finally, the quality of the silicone rubber usedshould be selected in light of the intended application of the finalproduct. In an especially preferred embodiment, silicone rubber paintRTV 118 (General Electric Co., Connecticut, USA) is used. In order toassist adhesion of the silicone rubber layer to certain materials, itmay be necessary to apply a conventional adhesive, such as a mineralspirit-based primer, prior to deposition of the silicone layer. In apreferred embodiment, the primer used is silicone rubber primer SS 4155(General Electric Co., Connecticut, USA). The micro-cupulated siliconerubber surfaces formed by the inventive method may be formed on orapplied to any suitable substrates. When applied to cell culturevessels, such as culture flasks or roller bottles, the textured siliconerubber surfaces have been found to produce greatly increased yields intissue culture processes. Such surfaces provide increased surface areafor cell attachment, as well as promoting or encouraging cellattachment. The increased surface area also enhances oxygen supply tothe surface. Thus, textured silicone layers according to the inventionmay be used in a variety of devices, particularly those where cellattachment is important.

In a third aspect of the invention, there is provided a method of makinga silicone rubber having a structure adapted for growth of cells orliving tissue substantially in accordance with the invention in itsfirst aspect, wherein the sacrificial filler is dispersed throughout thesilicone rubber precursor, and the structured silicone rubber issubstantially porous. In this aspect, the inventive method creates asystem of pores and channels throughout the silicone rubber structure.The pores of the silicone rubber provide sites of attachment for cellsor tissues, so that the cells or tissues may be substantially trappedwithin the resultant structure. This system of pores can also act as acapillary system, increasing oxygen and nutrient supply to the surfaceof the structure. In a preferred embodiment, the method of making aporous silicone rubber comprises mixing the biologically-acceptablesacrificial filler with the silicone rubber precursor, curing theresultant mixture at a temperature below 180° C., and removing thesacrificial filler, to form a porous silicone rubber. Preferably, thesilicone rubber is cured at a temperature between 100° C. and 175° C.,more preferably between 120° C. and 170° C., more preferably between140° C. and 160° C., and most preferably about 150° C. The siliconerubber precursors and the biologically acceptable sacrificial fillersthat can be used are essentially the same as described above in relationto the invention in its first or second aspects. In another embodiment,the resultant mixture may be shaped prior to curing, preferably bymoulding or extrusion. In a preferred embodiment, the average size ofthe pores formed is 1 μm-0.5 mm, preferably 10 μm to 0.2 mm, and morepreferably 50 to 150 μm in diameter. Preferably, the porous siliconerubber is cut to a desired size or shape. For example, the poroussilicone rubber may be cut in the form of small pellets that are capableof allowing cell growth within their pores but which can be readilyseparated from the culture medium by traditional separation methods,such as centrifugation or filtration. Silicone rubbers frequentlycontain innate fillers, such as fumed glass, which are added to producedesired viscosity, strength and other physical properties. The amount ofsacrificial filler that can be mixed into the silicone rubber and,therefore, the extent of the porosity achieved is inversely proportionalto the quantity of innate filler already present. Thus, a low viscositysilicone rubber containing small amounts of innate filler canaccommodate a greater packing density of sacrificial filler than a highviscosity silicone rubber containing high levels of innate filler togive it a thicker consistency. The viscosity of the silicone rubber isalso of importance when considering the manner in which the mixture isto be manipulated to give the end product. For example, if the mixtureis to be extruded, a low viscosity silicone rubber, although able tohold a greater amount of sacrificial filler, may not be suitable,because separation of the filler can occur, particularly if extrusionsof small cross-sections are required, as well as slumping of themixture, due to its low viscosity, which can result in distorted shapes.However, if the mixture is to be spread into a sheet or moulded, then alow viscosity silicone rubber may well be appropriate because, for thesame amount of filler, it is more easily manipulated. The greenstrength, i.e., the strength of the uncured silicone rubber precursormixture, is also a factor for consideration. Low viscosity siliconerubber, when packed with sacrificial filler, for example, exhibits verypoor green strength and is, thus, generally undesirable for extrusion.For extrusion applications, a very high viscosity silicone rubber wouldbe ideal in principle, but, as so little sacrificial filler can be mixedinto these materials, they are not usually a practical option.Therefore, a silicone rubber somewhere between the two must be chosen,such that sufficient innate filler can be included to maintain greenstrength but insufficient to be able to pack in sufficient sacrificialfiller. The cure regime of the silicone rubber must also be taken intoaccount. Where a rapid cure is required, for example, so as to maintainthe geometry of an extrusion, heat cure systems are often required.However, these systems must be tailored such that the heat process doesnot have a deleterious effect on the filler. It may also be necessary touse room temperature curing systems if the material needs to be bound toan additional substrate that is unable to withstand elevatedtemperature, such as a thermoplastic, for example, The physicalproperties of the cured silicone rubber must also be considered. Wheredurability is an important issue, such as in the formation of tubes orsheets, then a silicone rubber having high tensile strength must beused. However, such silicone rubbers tend to be of higher viscosity andcontain large amounts of innate filler and, hence, a compromise must befound. Where tensile strength is less of an issue, a low viscositysilicone rubber may be used, especially if there is no requirement forextrusion. Finally, the actual grade of silicone rubber is worthy ofnote. The final application of the material will determine the qualityof the silicone rubber to be used. For medical and implantableapplications, a high purity grade of material should be used, and,conversely, industrial grade silicone rubbers may be appropriate forapplications, such as waste water treatment. In some instances, it maybe desirable to include additives in the mixture in order to achievecertain characteristics, such as desired density, magnetic propertiesand the like. In the majority of cases, such additives would generallybe in powder form and the considerations needed to choose suitablematerials would be similar to those for the sacrificial filler. Forexample, if the silicone rubber is required to have an increaseddensity, a high mass powder would be added in small quantities to makethese adjustments and the choice of powder would follow criteria such asreactivity, toxicity and economics, etc. It has been found by thepresent invention that the use of certain sacrificial fillers can havean adverse effect on the resultant silicone rubber. For example, the useof sodium chloride can cause the silicone rubber to swell, dependingupon the conditions. In order to avoid, this, it is desirable to use asacrificial filler that does not cause swelling or adversely effect theresultant silicone rubber. Sodium bicarbonate has been found to beparticularly effective in satisfying such criteria, although a number ofother sacrificial fillers may be equally effective. If sodiumbicarbonate is to be used as a sacrificial filler, it decomposes and,therefore, “blows” the material at temperatures above approximately 180°C. Consequently, it is necessary to adapt the manufacturing process soas to avoid temperatures above 180° C., for example, by selectingsilicone rubbers, which cure at lower temperatures. Many of thealternative sacrificial fillers are toxic, leave toxic residues whendissolved, or are problematic at moderate temperatures required forworking with silicone rubber. The silicone rubbers formed using themethods in accordance with the first, second and third aspects of theinvention have properties that make them particularly well-suited foruse in a range of biomedical devices and apparatus.

In a fourth aspect of the invention, there is provided a culture chamberfor use in a method of culturing microbiological material, whichcomprises at least one gas-permeable wall or portion of a wall, and atextured interior growth surface arranged for contact with themicrobiological material being cultured. The general principles ofculturing cells in vitro are well-established in the field ofbiotechnology, with the term “cell culture” being usually understood torefer to both growth and maintenance of cells. In a preferredembodiment, the gas-permeable wall and the textured interior growthsurface are each formed from an organic polymer, optionally the sameorganic polymer. The gas-permeable wall or potion of a wall of theculture chamber may also provide the textured interior growth surface,such that cells may grow directly on a textured growth surface on thegas-permeable membrane, thus allowing high cell densities. In apreferred embodiment, the gas-permeable wall or portion of a wallcomprises a silicone rubber membrane. In an especially preferredembodiment, the textured interior growth surface is obtained orobtainable by a method according to the invention in its second aspect.Preferably, the culture chamber has at least one port extending betweenthe interior and the exterior of the chamber. More often, however, therewill be at least two ports, preferably including an inlet and an outletport. An additional septum port may also be provided, to reduce the riskof contamination when introducing various substances to the culturechamber. In an embodiment, at least one or both of the inlet and outletports are septum ports. In an especially preferred embodiment, theculture chamber is in the form of a flexible bag or envelope. A varietyof different apparatus is known for culture of cells in vitro. In recentyears, flexible culture bags have become increasingly popular, offeringa number of advantages over traditional cell culture apparatus, such asmulti-well plates, flasks, roller bottles and spinner flasks. Forexample, culture bags represent closed systems, thus reducing the riskof contamination, as well as taking up less storage and incubator space.In addition such culture bags can often be produced relativelyinexpensively, making them effectively disposable and reducing any needto sterilise them for re-use. In most tissue culture applications,aeration of the culture is essential in order to provide the cells withoxygen necessary for growth. In the past, methods such as sparging,surface aeration and medium perfusion have been used to increase oxygenavailability. However, such methods can cause cellular damage, therebyseverely limiting the efficiency of cell culture. Silicone rubbers havethe highest oxygen permeability of known polymers, and tubing ormembranes made from such materials are well-suited for use in cellculture, where they are able to provide improved diffusion of oxygen tothe cells. Silicone rubbers not only provides gas permeability(including oxygen and carbon dioxide) but also vapour transmission,structural integrity, resilience and temperature resistance, all ofwhich are desirable in cell and tissue culture. International patentapplication no. PCT/US96/20050 (Avecor Cardiovascular, Inc.) discloses acell culture bag formed from a plurality of thin, spaced, gas-permeablesilicone membranes, whose gas exchange rate is claimed to besignificantly higher than most conventional culture bags. However,although such bags may be capable of sustaining higher cell densitiesand cell viability, they are ultimately limited by the surface area ofthe bag. Moreover, the interior surfaces of such bags are smooth and,thus, provide poor cell attachment features, making them unsuitable forefficient cell culture of anchorage-dependent cells. Furthermore,certain “problem” cell types are unable to attach to the smooth interiorsurface of the bags. An elaborate (and seemingly expensive) method ofincreasing the surface area available for cell adhesion is described inU.S. Pat. No. 4,937,194 (Baxter International, Inc.), which discloses aflexible bag containing an internal cellular structure, such as ahoneycomb type structure with hexagonal channels passing through it,serving as adherent sites for cells being cultured. This document alsoproposes the use of microcarriers, such as small glass spheres or sodiumalginate, to increase the surface area available for cell adherenceinside the bag. There is a need, therefore, to overcome some of theaforementioned disadvantages. Accordingly, in an especially preferredembodiment of the invention in its fourth aspect, there is provided aculture chamber in the form of flexible bag or envelope. Such a culturebag provides an increased growth substrate surface area for cellattachment, as well as providing a growth substrate that will assistcell attachment. Moreover, the bag structure is simple and inexpensiveto manufacture. In a preferred embodiment, the bag is made from at leastone silicone rubber sheet that is coated with a silicone rubber layerhaving a rough or uneven micro-cupulated growth surface exhibiting aplurality of craters or crater-like depressions. Preferably, aroom-temperature vulcanising silicone rubber precursor is used, whilstthe sacrificial filler used to produce the textured surface ispreferably sodium chloride. The textured or micro-cupulated surface soformed significantly increases the surface area for cell attachment,thereby increasing the efficiency of cell culture. The micro-cupulatedsurface also assists attachment and growth of certain “problem” celltypes, such as, for instance, stromal cells necessary for stem cellexpansion processes. Stromal cells originating from bone require atextured surface on which to grow if their proliferation is to beoptimised. As already described above in relation to the culturechamber, the culture bag preferably also includes one or more ports,extending between the bag interior and bag exterior. Such ports may beused for introducing nutrient medium, taking samples, adding furtheringredients, etc. The ports should preferably have valves, locks or thelike, to avoid contamination of the big interior. In a preferredembodiment, the culture bag is provided with an inlet and an outlet portwith luer locks, and a septum port for taking samples or introducingsubstances into the bag. The ports are desirably positioned between thesealed edges of the culture bag. It has been found that the applicationof a textured surface to a culture bag wall in accordance with theinvention can result in the wall becoming opaque. In a preferredembodiment, therefore, the culture bag also includes at least oneportion of membrane to which no textured surface layer has been applied,this area serving to act as a transparent window, thus allowing a userto see inside the culture chamber. In a further embodiment, the culturechamber also includes a valve means, allowing the release of gases thatbuild up during cell growth and may form an air bubble inside the bag.The presence of a bubble within the chamber can prevent colonisation onthe surface area adjacent the bubble because the surface will not be incontact with the culture medium. Thus, the presence of a valve in theculture chamber wall helps to minimise the size of any gas bubbles,thereby allowing a larger surface area of the bag to remain in contactwith the nutrient medium and to be available for cell attachment. Almostcomplete colonisation on the interior chamber surface is, therefore,possible, increasing the efficiency of the culture chamber. Desirably,the valve comprises a filter means, allowing gases to diffuse out of thechamber but preventing microbial contamination. In a preferredembodiment, the valve means comprises one or more layers of ahydrophobic material, such as a hydrophobic PTFE membrane, preferablyhaving a thickness of around 0.25 mm and a porosity of 0.2 microns.However, other suitable forms of valves means will also be apparent tothose skilled in the art. The growth surface of the culture chamber orculture bag may be treated to further enhance cell adhesion, for exampleby charging the surface by bombardment with electrons. It is alsopossible to modify the free —OH groups of the silicone rubber surface toencourage attachment of various chemical moieties, in the manner alreadydescribed in relation to the invention in its third aspect.Alternatively, cell attachment to the growth surface of the culturechamber may be promoted by adapting the size of the micro-cupules ordepressions to the specific requirements of the cells to be cultured. Ina preferred embodiment, the culture chamber further comprises a secondchamber separated from the first chamber by means of a semi-permeablemembrane. The second chamber preferably has an access means separatefrom that of the first chamber.

In a fifth aspect of the invention, there is provided an apparatuscomprising a plurality of culture chambers according to the invention inits fourth aspect, for use in a method of culturing microbiologicalmaterial. In an embodiment, the inlets of the culture chambers areinterconnected and the outlets of the culture chambers areinterconnected. In a preferred embodiment, the apparatus has at leastone further chamber(s) having a semi-permeable wall that is positionedwithin each culture chamber, each semi-permeable chamber(s) having aninlet that is interconnected with the inlet of any other semi-permeablechambers and having an outlet that is interconnected with the outlet ofany other semi-permeable chambers. In a preferred application,anchorage-dependent stromal cells are grown on the textured surface ofthe culture chamber(s), and anchorage-independent stem cells are theninoculated into the culture chamber(s), to allow proliferation of thestem cells. Preferably, the apparatus is a bio-reactor. The bio-reactoris particularly applicable to the bio-processing of liquors containingparticular matter, such as blood cells or cell debris. Conventionally,bio-reactors are normally closed systems and, as such, have thedisadvantages of relatively low productivity and efficiency. Oneparticular drawback is the limited volume of oxygen available forreaction in such closed systems. Moreover, such systems not normallysuitable for the processing of liquors containing particular matter,such as whole blood. The bio-reactor according to the invention does notsuffer from the aforementioned problems because it comprises oxygenpermeable walls, and a textured surface of silicone rubber to assist thegrowth process of the bio-substances. Thus, the desired product may besubsequently generated in a continuous process by a passage of liquidnutrient medium over the bio-substances. In a preferred embodiment, amethod of carrying out a bio-processing operation in a culture chamberor an apparatus comprises attaching cells for performing thebio-processing function to the textured surface of the culturechamber(s), introducing liquor to be processed into the culturechamber(s) via an inlet and collecting the processed liquor at an outletfrom the culture chamber(s). Preferably, the spent medium includingcellular by-products is removed from the culture chamber(s), and freshnutrient medium is passed through a semi-permeable chamber(s) locatedwithin the culture chamber(s), so to allow fresh medium to diffusethrough the semi-permeable membrane into the culture chamber(s).Preferably, the nutrient medium is passed through the semi-permeablechamber in the opposite direction to that in which the liquor or spentmedium is passed through the culture chamber. This has the advantagethat cells growing in those areas of the culture chamber having the mostheavily depleted medium are contacted with fresh medium first. Thenutrient medium may be recycled. In a preferred embodiment, theapparatus is filled with liquid medium, which has first been inoculatedwith a desired cell line. The assembly of reactor tubes may then bearranged to be rotated or agitated, for example, using machinery such asthat employed for conventional roller bottles. Rotation may be continueduntil cell confluence is obtained, as evidenced by the levelling of therate of glucose uptake. The inner surfaces of the reactor tubes are,therefore, extensively coated with the cells at this stage. Ifappropriate, rotation may be interrupted for replacement of the mediumin the reactor. The reactor tubes can then be removed from the rollersand connected to a suitable media reservoir. A continuous stream ofliquid nutrient medium may be arranged to pass through the reactorenvelopes, the product being harvested at the outlet. During thisprocedure, it is desirable to provide an airflow over the reactor, toassist oxygenation. In a preferred embodiment, the apparatus isespecially adapted for bio-processing of liquors containing particulatematter, such as blood cells or cell debris. The continuous flow systemaccording to the invention is especially applicable to the processing ofwhole blood, for example, in an artificial extra corporeal organsubstituting or supporting the functions of the human liver.Advantageously, the system obviates the need of separating theparticulate matter prior to processing and then having to reunite theconstituents. It is also envisaged that the culture chambers andapparatus according to the invention may have other medicalapplications, such as for expansion of other primary cell types, or foruse as an ex vivo model for drug metabolism if colonised withhepatocytes and the like. In another embodiment, the culture chambersfurther include semi-permeable chambers positioned within themselves,such as, for example, semi-permeable chambers made of cellulose acetate.These semi-permeable chambers are arranged to be separately connected tocommon inlets and outlets at their respective ends. In this embodiment,the bio-processing operation involves the following procedures. First,the cells grown to perform the bio-processing function are attached tothe textured surface of the culture chamber. The culture medium is thenremoved from said culture chambers. Next, the nutrient medium is passedthrough the semi-permeable chambers, introduced from a reservoir throughthe inlet at one end of the semi-permeable chambers, issuing at theoutlet on the opposing end. If desired, the medium may be recycled fromthe outlet, to return again to the inlet of the chambers. The liquid tobe processed, such as blood, for example, is then arranged to flowthrough the culture chambers, the textured interior surface of which arenow coated with cells. The liquor is introduced for this purpose at theinlet of the culture chambers, formerly serving at the medium inlet, andissuing at the outlet on the opposing end. The liquor is preferablypassed through the culture chambers in opposing direction to that of thenutrient medium. During this procedure, nutrients from the medium passthrough the semi-permeable chambers, traversing the stream of liquor, tofeed the cells adhering to the coating of the culture chambers. At thesame time, the semi-permeable chambers also perform the function ofcleansing the liquor of waste materials, such as ammonia or urea, etc.The treated liquor is finally collected at the outlet of the culturechambers. The productivity and efficiency of the growth process,especially in the case of anchorage dependent cells, can besubstantially enhanced using the bio-reactors according to theinvention, especially when compared with conventional reaction vesselsthat do not utilise oxygen permeable containers and, thus, cannotsustain cell growth process in the manner permitted by the invention.

In a sixth aspect of the invention, there is provided a well for use ina method of culturing microbiological material having at least one walldefining the well, at least a portion of the wall being gas-permeable,to enhance oxygen supply to the well, and at least a portion of theinterior surface of the wall being textured, to increase surface areaand to enhance cell adherence. In a preferred embodiment, thegas-permeable portion of the wall and the textured portion of the wallare positioned at or near to the base of the well. In anotherembodiment, the gas-permeable portion of the wall comprises agas-permeable membrane, preferably formed of silicone rubber. Themembrane preferably has a textured surface facing the interior of thewell, to increase available surface area and to facilitate cellularattachment. In a preferred embodiment, the textured surface hascrater-like depressions or micro-cupules and is preferably a texturedsilicon rubber layer made by a method according to the invention in itssecond aspect. In a further embodiment, at least a portion of theinterior surface of the wall comprises porous silicone rubber inaccordance with the invention in its third aspect, preferably near thebase of the well. In an especially preferred embodiment, the poroussilicone rubber is provided with a textured silicone rubber layer whichserves to form the interior surface of the well. Such wells areparticularly useful in cellular assays which require the cells to remainsubstantially trapped in the wells, during a succession of stepsinvolving washing or treatment with various reagents.

In a seventh aspect of the invention, there is provided a microtitreplate having at least one well according to the invention in its sixthaspect. The wells help to increase both the quantity of cells that canbe grown in a microtitre well of a given size, as well as theirmetabolic activity. Preferably, the microtitre plate has at least onewell having a wall at least a portion of which comprises a texturedsilicone rubber surface in accordance with the invention in its secondaspect. As microtitre wells become increasingly minimized in size, thenumber of cells that can be grown in each well, for example, for drugmetabolism studies, is also reduced because of the decrease in availablegrowth surface area. Moreover, those cells that can be grown are alsostarved of oxygen due to the decrease in gassing surface to volumeratio. The microtitre plate according to the invention helps to eleviatethis problem by firstly increasing the available surface area with thetextured surface and secondly allowing the cells to be gassed from belowthe second membrane.

In an eighth aspect of the invention, there is provided an implantdevice comprising a cell support structure having a coating with atextured surface, to promote anchorage of the implant by cell attachmentand ingrowth by surrounding tissue upon implant. Preferably, thetextured surface has crater-like depressions or micro-cupules. In anespecially preferred embodiment, the coating comprises textured siliconerubber, preferably manufactured according to the invention in its secondaspect. The implant devices according to the invention may take manydifferent forms, such as, for example, a heart valve, a sternum implant,or a reconstructed calf ligament. The textured surface on the implantsacts as an anchor for tissue in-growth. Thus, the textured implant canhelp to prevent migration of larger implants or to promote a securebond, where the interface with the implant and the surrounding tissue iscritical. It has been found that the textured surfaces according to theinvention have the further advantage of helping to reduce the formationof capsule-type scar tissue following implantation.

In a ninth aspect of the invention, there is provided a substrate forgrowth of skin grafts in vitro comprising a flexible membrane having atextured surface. A major problem associated with the growth of skin exvivo is that, when it is grown on a rigid or solid surface, the skintends to be bristle and does not have the opportunity to “learn” to beflexible. In addition, the undersurface of the skin tends to be smoothand scar-like, which makes it difficult for the skin graft to take. Theflexible membrane used in the inventive substrate helps to prevent theskin graft from becoming brittle, whilst the textured surface increasesthe surface area available for cell adhesion, promotes cell adhesion andhelps to gives the skin a rough surface, so as to enhance “taking” ofthe graft on transplant. In an embodiment, the flexible membrane isgas-permeable, preferably comprising a material such as silicone rubber.In a preferred embodiment, the textured surface has crater-likedepressions or micro-cupules. Preferably, the textured surface is formedof silicone rubber manufactured according to the invention in its secondaspect. The textured surface not only provides a greater surface areafor cell growth, but also allows a degree of ingrowth into the siliconerubber in small areas, so that upon removal from the growth surface, theskin undersurface will be textured, assisting the taking process of thegraft. In addition, the high oxygen permeability of the silicone rubberwould assist in promoting the metabolic activity of he growing graft.

In a tenth aspect of the invention, there is provided a tissue supportstructure for use in a method of culturing tissue or cellularagglomerates, which comprises a biocompatible material having aninternal system of pores, the pores promoting cell attachment andanchorage and oxygen supply to the tissue. Microparticles of organsgrown ex vivo have many applications in the drug development industry.However, conventional support substrates for tissues or tissue fragmentsgrown ex vivo are severely limited as to the size of the tissueagglomerates that may be grow. The need to provide oxygen and nutrientsto the centre of a three dimensional tissue mass has been widelyrecognised by those skilled in the art and has been addressed in anumber of different ways, all of which involve complex and expensivesupport structures having specific structural features for gas andnutrient supply. The tissue support structures according to theinvention have a system of pores and channels within the porousstructure that is capable of mimicking a biological capillary system,delivering oxygen directly to the centre of the tissue growing on thestructure. This allows much larger agglomerates to be formed whileavoiding necrosis and apoptosis. In a preferred embodiment, the porousmaterial is provided with small, fine bore tubes. In another embodiment,the shape of the porous material may be adapted so as to engineer theshape of the resultant tissue. In a preferred embodiment, the porousmaterial comprises porous silicone rubber, preferably made according tothe invention in its third aspect. In an especially preferredembodiment, the tissue support structure comprises porous siliconerubber having small, fine bore tubes. In this case, the silicone rubbermay be provided with a porous and micro-capillary structure by usingfinely ground sacrificial filler and long thin needles of crystallinesacrificial filler in admixture in the process according to theinvention in its third aspect, the ground filler serving to provide theporous superstructure and the needles of filler serving to provide themicro-capillaries. However, artificial capillaries may also beintroduced into the porous silicone rubber by other methods, such astrapping gases in the setting silicone rubber, mechanical disruption,laser ablation, etc.

In an eleventh aspect of the invention, there is provided an apparatusfor culturing tissue or cellular agglomerates comprising a tissuesupport structure according to the invention in its tenth aspect and agas-permeable membrane, to enhance oxygen supply to the system of poresand channels within the porous material, and therefore to the tissue.Preferably, the gas-permeable membrane is attached to the porousmaterial. In a preferred embodiment, the gas-permeable membrane is asilicone rubber, preferably made in accordance with the invention in itsthird aspect. Preferably, the porous material is attached to thegas-permeable membrane using a gas-permeable adhesive, such as asilicone rubber adhesive. In a preferred embodiment the plurality oftissue support structures are arranged in close proximity to oneanother, so as to allow fusion between tissue or cell masses growing oneach structure, to create larger tissue or cellular agglomerates. It isenvisaged that tissue grown on this type of structure could reachmacro-dimensions being fed with oxygen via diffusion through solidthreads attached to tubes through which oxygen would be passed.Preferably, the support structure is in the form of a pillar, thedimension of which are approximately 0.25 mm×2 mm.

In a twelfth aspect of the invention, there is provided an artificialimplant formed from a material having an increased system of pores, thepores promoting cell attachment and anchorage and oxygen supply to thecells on the implant surface. The pores throughout the structure allow adegree of ingrowth and anchorage of the cells, as well as a pathway forsupply of oxygen to the cells on the surface. In a preferred embodiment,the porous material comprises porous silicone rubber, preferably madeaccording to the third aspect of the invention. In a preferredembodiment, the artificial implant is adapted for use as a cartilageimplant. In such applications, the porous silicone rubber is carefullyselected to contain the necessary degree of biologically inert filler togive it the required degree of elasticity and/or flexibility of thecartilage. Preferably, the porous material is seeded in vitro withchondrocites, to form a layer of cartilage over the implant. Such animplant may be used for replacing eroded joints and the porous siliconestructure may be moulded to conform to the shape of the bone it is toprotect. Prior to the present invention, cartilage intended for suchpurposes was grown in vitro in a flat single layer on culture plates andwas then placed over the eroded bone. The principle disadvantage of sucha method is that the cartilage so grown is in a flat form and does notreadily accommodate to the contours of the bone to be protected. Theimplants according to the invention do not suffer from such adisadvantage and, thus, may be used to provide “spare parts” for surgeryin the human body. In another preferred embodiment, the porous materialof the cartilage implant could be moulded into the shape of a nasalbridge, or an ear. This type of permanent synthetic bio-compatibleimplant offers both support and a degree of permanent protection to thecartilage structure.

In a thirteenth aspect of the invention, there is provided an artificialimplant according to the invention in its twelfth aspect in the form ofa vascular graft. Conventional vascular grafts often suffer an adversefate because their base material has incompatible physical properties tothose of the native tissue. In a preferred embodiment, the vasculargraft of the present invention comprises a hollow tube made from porousmaterial, preferably porous silicone rubber. In an embodiment, theinterior surface allows cell adhesion, and preferably endothelial cellsare grown on the interior surface of the graft. In another embodiment,the exterior surface also allows cell adhesion, and preferably smoothmuscle cells are grown on the exterior surface of the graft. In apreferred embodiment, one or both surfaces of the graft are additionallyroughened to enhance cell attachment, preferably by providing the graftwith textured silicone rubber surface. The elastic, compression andoxygen transport characteristics of porous silicone rubber closely mimicthose of living tissue and, thus, help permit common problems, such asstenosis, to be overcome. A further advantage associated with thevascular grafts according to the invention are that these produce alamina flow and not the turbulent flow associated with rigid syntheticgrafts, hence helping to minimizing the problems of thrombosis. Due tothe chemical properties of silicone rubbers, such vascular grafts wouldalso be resealable, which would be advantageous for patients requiringrepeated vascular access, for example, patients suffering from renaldisease and undergoing long-term kidney dialysis.

In a fourteenth aspect of the invention, there is provided a cellimplant means comprising a porous material for retention of cells to beimplanted, the pores promoting cell attachment and anchorage and oxygensupply to the cells, and a protective means to shield the cells fromimmune attack after implant. In an embodiment, the porous materialcomprises silicone rubber, preferably made by the method according tothe invention in its third aspect. In a preferred embodiment, theprotective means desirably comprises a semi-permeable membrane formingan envelope around the porous material. In an especially preferredembodiment, the cell implant means is adapted for use as an endocrineimplant. The porous material is seeded in vitro with endocrine cells,such as islets of Langerhans cells. The development of fully-functionalendocrine implants, especially of the islets of Langerhansinsulin-secreting cells, has long been a target for clinical research.However, expanding islet cells has proved extremely challenging, becauseit is difficult to make them proliferate in culture. Islet cells fromfoetal tissue have been proliferated in culture but permanently losefunction over time. The endocrine implants according to the presentinvention should help to overcome such difficulties. On implantation ofthe endocrine implant, the required hormone is released through thesemi-permeable membrane, whilst this membrane also acts as a barrier tothe body's own defences against the foreign cells. Regulation of thehormones released can occur naturally, as feedback control molecules areable to pass through the semi-permeable membrane and communicate withthe endocrine cells directly.

In a fifteenth aspect of the invention, there is provided a drugdelivery system comprising a porous material whose pores have beenimpregnated or saturated with a drug for delivery. Preferably, the drugdelivery is suitable for implantation into a human or animal body. In apreferred embodiment, the drug is present in admixture with at least onesustained release ingredient. In an especially preferred embodiment, theporous material comprises a porous silicone rubber, preferably made by amethod according to the invention in its third aspect. Many drugs existas small molecules which are capable of diffusing readily throughsilicone rubber. Such drugs can be incorporated into the porous siliconerubber, which acts as a drug delivery system. Advantageously, the porousnature of the silicone rubber means that it is capable of exposing alarge surface area to bodily fluids for a relatively small implant.Moreover, the synthetic nature of the silicone rubber means that thesystem is less likely to be rejected by the body when implanted. Inaddition, the material will not biodegrade as with many of the currentdevices, making it possible to explant the spent device for analysis andmonitoring purposes if required.

In a sixteenth aspect of the invention, there is provided a filtrationmedia comprising porous silicone rubber, for use in separations.Preferably, the porous silicone rubber is made according to theinvention in its third aspect. In a preferred embodiment, the pores ofthe silicone rubber are of sub-micron size, preferably in the order of 1nm-10 μm, more preferably in the order of 10 nm-5 μm, and morepreferably about 0.1-0.5 μm. The filtration media may be used inmagnetic separation and, in such a case, the porous silicone rubberpreferably includes magnetic additives. The filtration media may also beused in expanded bed adsorption and, for this application, the poroussilicone rubber is preferably in particulate form. The filtration mediamay be for use in static inline filtration, for which the poroussilicone rubber is preferably in the form of sheets or tubes. In apreferred embodiment, the filtration media comprises porous siliconerubber in the form or annular discs. Preferably, porous silicone rubberwith a sub-micron pore size is used. In particulate form, the filtrationmedia according to the invention is highly suited for use in theburgeoning market of expanded bed absorption technology. This is becauseporous silicone rubber can be easily modified to have the appropriatedensity and, due to its elastic nature, can be used in whole broth orcontinuous processes over protracted periods of time. After primaryprocessing, the filtration media can be made receptive to all commonmoieties used in affinity chromatography processes. In addition, thefiltration media according to the invention can also easily be mademagnetic, so that it can be easily separated from a whole broth systemusing magnetic separation.

In a seventeenth aspect of the invention, there is provided a cellcryopreservation system comprising a porous material for absorbing cellculture into the internal system of pores and a container suitable forstorage in liquid nitrogen. In a preferred embodiment, the porousmaterial comprises porous silicone rubber, preferably porous siliconerubber made in accordance with the invention in its third aspect. Thecontainer preferably comprises releasable sealing means. In analternative embodiment, the container is a syringe-type plunger. In suchan embodiment, a number of cylindrical particles of porous siliconerubber may be placed in a tube fitted with a syringe type plunger. Anoperator could then suck up the required culture to saturate the poroussilicone rubber particles and then store the device in liquid nitrogen.Upon retrieval, the operator then has a number of porous silicone rubberparticles containing the same culture that can be used for severalinoculums.

In an eighteenth aspect of the invention, there is provided an electrodecomprising a porous material having electrically conductive particlesdispersed therein. In a preferred embodiment, the porous materialcomprises porous silicone rubber, preferably a porous silicone rubbermade in accordance with the invention in its third aspect. Preferably,the conductive particles are metal or carbon powders. In anotherembodiment, the porosity of the electrode material promotes adherence ofmicroorganisms to the electrode surface. In an especially preferredembodiment, the microorganisms are capable of digesting waste, such thatthe electrodes may be used in the treatment of sewage and in similarapplications. In another embodiment, the electrode forms part of anelectrode system comprising a plurality of electrodes immersed in aliquid electrolyte and connected to an electric circuit. As inconventional electrolytic systems, in use, two electrodes (a cathode andan anode) are immersed in the liquid electrolyte, are connected to anelectric circuit with a potential being applied between them. In specialapplications, electrolytic baths may comprise a plurality of electrodes.The porous silicone rubber electrodes have a number of advantageousfeatures, including a large surface area and, hence, a high electricalcapacity, robustness, inertness and resilience (aided by some degree ofelasticity). Such characteristics are particularly important in therelatively hostile chemical and physical environment of agitated liquidelectrolytic cells. Furthermore, the porous silicone rubber alsoprovides a favourable surface for the growth of micro-organisms makingsuch electrodes particularly suitable for special uses in waterpurification and sewage treatment applications. Traditionally, suchwater treatments normally comprise the functions of converting (a)carbonaceous material to carbon dioxide and water, (b) nitrites tonitrates, and (c) nitrates to atmospheric nitrogen, all three functionsrelying on the actions of micro-organisms. Among difficulties associatedwith conventional procedures are those of providing an adequate streamof oxygen through the sewage to maintain the micro-organism activity.This usually requires agitation of the liquid using mechanical stirrers,while passing a stream of oxygen through the sludge in the case offunctions (a) and (b) and providing a safe reducing atmosphere for (c).Using porous silicone rubber electrodes according to the invention in,for example sewage treatment, advantages are achieved in respect ofgreater output efficiency, enhanced reliability owing to the absence ofmoving parts in the system, as well as lower operating costs. By usingthe porous silicone rubber electrodes, an oxygen stream is applied atthe anode to pass through the sewage, allowing micro-organisms to effectthe reactions (a) and (b), while an enhanced level of hydrogen at thecathode aids the conversion by micro-organisms of (c).

In a nineteenth aspect of the invention, there is provided a wounddressing comprising a first layer of a porous gel and a second layer ofa carrier gel. In a preferred embodiment, the porous gel layer comprisesa porous silicone rubber gel, preferably made by a method according tothe invention in its third aspect. The carrier gel layer may alsocomprise a silicone rubber gel. Preferably, the carrier gel is appliedto a supportive structure, such as a Dacron® mesh. In a preferredembodiment, the porous gel layer is infused with a drug for delivery tothe wound, such as a growth-promoting drug or an antibiotic. Thesilicone rubber wound dressing according to the invention also helps tocontrol scar formation by leaching low molecular weight silicones intothe wound, a technology already employed in the field. The siliconerubber wound dressing has the added advantage of increasing the contactarea with fluids from the wound, thereby improving leaching and allowinggreater oxygen transport to the site, whilst maintaining asepsis.Moreover, the dressing permits drugs infused into the porous structureto leach into the wound over a prolonged period of time to aid thehealing process.

In a twenteeth aspect of the invention, there is provided a clinicalswab comprising a porous material, the pores increasing the surface areaof the swab and promoting oxygen transport to the swab surface. In apreferred embodiment, the porous material comprises porous siliconerubber, preferably made by a method according to the invention in itsthird aspect. In a further embodiment, the porous material contains aradio-opaque additive, such as barium sulphate. This allows any lostswabs to be easily traced and then removed. In another embodiment, theporous material is infused with a drug. The swab is preferably attachedto the end of a stick made, for example, of wood or plastic. The swabaccording to the invention has a number of advantages over theconventional swabs. The porous silicone rubber is oxygen permeable. Thesilicone rubber is also non-limiting, reducing the risk of debris beingleft behind after use. The silicone rubber is also better attached tothe stick than cotton wool in conventional swabs. The silicone isfurthermore chemically very stable and will also allow microorganisms toadhere to the swab surface.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the invention may be better understood, examples of thevarious aspects will now be described, by way of illustration only andwith reference to the accompanying drawings, wherein:

FIGS. 1, 2 and 3 show successive steps of the manufacturing process inaccordance with the second aspect of the invention;

FIG. 4 shows a cross-sectional view of three-dimensional porous siliconerubber in accordance with the third aspect of the invention;

FIG. 5 is a schematic plan view of a culture bag in accordance with thefourth aspect of the invention;

FIG. 6 is a schematic cross-sectional view of the culture bag of FIG. 5;

FIG. 7 is a cross-sectional view of a membrane wall of the culture bagof FIGS. 5 and 6;

FIG. 8 is an exploded view of the valve of the culture bag of FIGS. 5and 7;

FIG. 9 is a diagrammatic illustration of a bio-reactor apparatusaccording to the fifth aspect of the invention;

FIG. 10 is a silicone rubber tube from the bio-reactor of FIG. 9;

FIG. 11 is a cross-sectional side view of a bio-reactor apparatus withdialysis tubes;

FIG. 12 shows plan view of a microtitre plate according to the sixthaspect of the invention;

FIG. 13 shows a cross-sectional view of the plate of FIG. 12 along lineA-A′;

FIG. 14 shows a schematic artificial capillary system, in accordancewith the ninth aspect of the invention; and

FIG. 15 shows a part cross-sectional view of an endocrine implantaccording to the thirteenth aspect of the invention.

DETAILED DESCRIPTION OF THE INVENTION

FIGS. 1, 2 and 3 show successive steps of the manufacturing process inaccordance with the second aspect of the invention. In FIG. 1, thesurface of a substrate 10 is coated with a layer of uncured siliconerubber precursor 11. In FIG. 2, a sacrificial filler 12, such as sodiumchloride, is applied to the silicone rubber layer 11 whilst the latteris still tacky, the sodium chloride 12 becoming adhered to and partiallyembedded in the silicone rubber layer. Any excess sodium chloride 12that is not adhered to the silicone rubber layer 11 is removed and thesilicone rubber layer 12 is allowed to cure. Once the silicone rubberlayer 11 has been cured, the sodium chloride 12 is dissolved in asolvent, such as water, leaving craters or micro-cupules 13 forming atextured surface structure 14 as shown in FIG. 3.

In FIG. 4, a porous silicone rubber article 70 has a textured exteriorsurface with craters 71 and pores 72 within the body of the siliconerubber article 70, forming porous channels throughout the threedimensional structure. The porous silicone rubber article 70 is madefrom GE Silicone's LIM 6070-D2 (part A & B) or McGhan NuSil's MED 4970(part A & B), to form the silicone rubber and J. Astley & Sons FoodGrade NaHCO₃ (sodium bicarbonate) as sacrificial filler. Stainless steelpowder (MBC Metal Powders Ltd 316L SS fines 325 mesh) is also added fora high density silicone rubber product. The sodium bicarbonate is mixedwith each of parts A and B of the silicone rubber separately, at a ratioof 3:1 w/w. The mixing is carried out using a conventional Z-blademixer, although other mixer types may be used, or mixing may even beperformed by hand. Stainless steel powder is added to a level to givethe desired density (although other high mass powders, such as titaniumoxide, can also be used). Once mixed with the sodium bicarbonate, partsA & B are stored separately in a cool place for further processing. Thecomponents must be kept apart as one contains the accelerator and theother the catalyst that will cause curing. If cross-contamination of theparts occurs, the material will start to cure. When ready to cure thematerial into the required shape, parts A & B are mixed together on atwo roll mill for 15 to 20 minutes to ensure complete mixing. Theresultant mixture is then fed into a cold head extruder and extrudedthrough a die of the appropriate shape. The resultant extrudate ispicked up by a heat resistant conveyor and passed through a hot box setto such a temperature that the extrudate itself is heated to 175° C.This facilitates the cure of the material without allowing the sodiumbicarbonate to decompose and hence “blow” the material. Depending uponthe geometry of the extrudate, it is passed through either a rotarycutter (for small cross-sections) or a reciprocating cutter (for largergeometries) and chopped into the appropriate particulate shape. This“preform” is the stored in a dry place until further processing isrequired. When required, the material is boiled in at least a five-timesexcess of pyrogen-free water for one hour. This process is repeated fouror five times or until no further traces of sodium bicarbonate areevident, as indicated by the pH of the water. The material is thenfinally rinsed in pyrogen-free water, bottled in an excess of the sameand autoclaved to facilitate sterile storage. The material is now in aform ready for sale as a stand alone support matrix.

In a further example, the porous silicone rubber article 70 is made GESilicone's RTV (room temperature vulcanising) 615 (part A & B), as thesilicone rubber material, and J. Astley & Sons Food Grade NaHCO₃ (sodiumbicarbonate), as the sacrificial filler. For a high density siliconeproduct, iron oxide (magnetic precipitate) from Fishers ScientificProducts is used. The sodium bicarbonate is wet milled under xyleneusing a Biaton bead mill to a particle size of 0.1 to 0.4 μm. This rangecan be further narrowed by separation in a Malvern® particle sizer.Using these methods, a whole range of particle sizes and distributionscan be achieved. The sodium bicarbonate is mixed with each of parts Aand B of the silicone rubber separately, at a ratio of 3:1 w/w. Themixing is carried out using a conventional Z-blade mixer, although othermixer types may be used, or mixing may even be performed by hand. If thedensity is to be increased, the iron oxide is added to a level to givethe desired density. Other high mass powders such as titanium oxide canalso be used. Further weighting or magnetic moieties may also be mixedin, if required. Once mixed with the sodium bicarbonate, the parts A & Bare stored separately in a cool place for further processing. When readyto cure the material into the required shape, parts A & B are mixedtogether on a two roll mill for 15 to 20 minutes to ensure completemixing. Again other apparatus could be used. The resultant mixture isthen fed into a cold head extruder and extruded through the open scrolland collected as ingots on trays. The ingots are then cured at 150° C.in a standard convection oven. The ingots are then ground in a mill tothe required size and can again be sized using a Malvern® particle sizerif required. This “preform” is the stored in a dry place until furtherprocessing is required. When required, the material is boiled in atleast a five-times excess of pyrogen-free water for one hour. Thisprocess is repeated four or five times or until no further traces ofsodium bicarbonate are evident, as indicated by the pH of the water. Thematerial is then finally rinsed in pyrogen-free water, bottled in anexcess of the same and autoclaved to facilitate sterile storage. Thisproduct is biocompatible, it has pores in a very well defined size rangeand of an amorphous geometry.

In FIGS. 5 and 6, a culture bag 20 comprises two membranes 28 joined attheir outer edges 27, each membrane 28 having a textured (interior)surface 26. Inlet and outlet ports 23 extend between the inside and theoutside of the bag, each port 23 being provided with a valve 24. Adegassing valve 22 is provided in the centre of one of the membranes 28,this membrane 28 being uppermost when the bag 20 is in use. In FIG. 7,each bag membrane 28 is prepared by covering the edges 27 of a smoothsilicone rubber sheet 25 with a mask (not shown) and applying a layer ofroom-temperature vulcanising liquid silicone rubber to the exposedcentral portion of the sheet. Next, vacuum-dried salt is sprinkled overthe layer of liquid silicone rubber so that it is uniformly covered. Theliquid silicone rubber is then cured and the salt is washed out,producing a membrane 28 with a cratered or micro-cupulated surface 26.In FIG. 8, a degassing valve is formed by first cutting a hole 31 out ofthe centre of one of the membranes 28, over which the valve will beplaced. A washer 29 made of uncured silicone rubber is positioned aroundthe hole 31 on the smooth (outer) face of the membrane 28. A hydrophobicPTFE membrane 30 with 0.2 μm pores and a thickness of 0.25 mm is laidover the washer 29, and a second washer 29 is placed on top. This isthen repeated with a second PTFE membrane 30 and a third washer 29. Whenthe bag 20 is to be assembled, the two silicone rubber membranes 28 arelaid on top of one another, with the rough surfaces together. Twolengths of tubing for the inlet and outlet ports 23 are placed betweenthe silicone rubber membranes 28, protruding slightly into the rougharea. The ports 23 are provided with valves 24. Next, room temperaturevulcanising silicone rubber is applied to the untreated, smooth edges 27of the silicone membranes 28, along which the membranes 28 are to bejoined to form a bag configuration 20. Uncured silicone rubber isapplied around the tubing where it lies adjacent to the smooth edges ofthe membranes 28. The constituents of the culture bag 20 so arranged arethen welded or glued together using elevated temperatures and pressure.The edges of the silicone membranes 28 are sealed to form a bag 20, thedegassing valve is formed from the layers of washers 29 and PTFEmembrane 30, and the tubing for the ports 23 becomes integrated into thebag structure 20.

In FIG. 9, a bio-reactor apparatus comprises two reactor tubes 40 (inpractice, a larger number, such as seven or eight tubes, is generallypreferred). Each reactor tube 40 carries an internal coating of texturedsilicone rubber 41. In use, in order to grow the cells on interiorsurfaces 41 of tubes 40, medium carrying cell lines is introducedthrough an inlet 43. Reactor tubes 40 are interconnected throughdistributors 42. One or more strengthening members 45 ensure rigidity ofthe assembly. The assembly is rotated on rollers (not shown), followedby evacuation of the liquid and subsequent passage of nutrient mediumover the cells. The medium is introduced through the inlet 43 and issuesfrom the outlet 44. The product is finally collected at the outlet 44.In FIG. 10, the reactor comprises a non-porous silicone rubber tube 40carrying an internal coating of textured silicone rubber 41. In FIG. 11,dialysis tubes 51 are co-axially positioned within the reactor tubes 40.Cells are grown in the annular space 52 by the passage via introductionof medium comprising the cell line through the inlet 47. After removalof the liquid from the annular space through the outlet 48, the nutrientmedium is passed through the dialysis tubes 51 via medium inlet 49,issuing at outlet 50. At the same time, the liquor to undergo thebio-reaction is passed through the reactor tubes via inlet 47, forcollection at outlet 48.

In FIGS. 12 and 13, a standard microtitre plate 60 has wells 61 withoutbase walls (either conventional microtitre plates are used and the basewalls of the wells removed, or a microtitre plate is produced withoutany base walls). A non-porous silicone membrane 62 is attached to thebottom of the wells, the membrane comprises a silicone rubber sheet 63having a coating with a textured surface 64 facing the area defined bythe wells.

In FIG. 14, a tissue growth support structure comprises a tissue mass83, such as HT-29 (intestinal carcinoma) cells grown on pillars ofporous silicone rubber 81, the pores acting as a capillary system,supplying oxygen to the cells in the centre of the tissue mass 83. Thepillars 81 are attached to a gassing membrane 80 in a bio-reactorconfiguration, using gas permeable silicone rubber adhesive 82. Theoxygen diffuses through the gassing membrane 80 and through the systemof pores and channels to reach the tissue agglomerate 83. The tissuegrowth support structure permits much higher densities of HT-29 cellsthan conventional systems.

In FIG. 15, islet of Langerhans cells are immobilized within a bio-wafer90 consisting of a disc of porous silicone rubber 92, in which the isletcells are attached, sandwiched between semi-permeable membrane layers91, which allow insulin out but stop host immune cells from attackingand destroying the transplanted islet cells.

1. A porous silicone rubber article having a structure adapted forgrowth of cells or living tissue obtained by a method comprising mixinga biologically acceptable sacrificial filler with a silicone rubberprecursor, curing the resultant mixture at a temperature below 180° C.,and removing the sacrificial filler by dissolution to form a poroussilicone rubber article, said sacrificial filler is an inorganic saltthat has been ground, and said inorganic salt is selected from the groupconsisting of metal halides, metal carbonates and metal bicarbonates. 2.A biomedical device comprising a porous silicone rubber article asclaimed in claim
 1. 3. A method of making a silicone rubber articlehaving a structure adapted for growth of cells or living tissue, whichcomprises mixing a biologically acceptable sacrificial filler with asilicone rubber precursor, curing the resultant mixture at a temperaturebelow 180° C., and removing the sacrificial filler by dissolution toform a porous silicone rubber, said sacrificial filler is an inorganicsalt that has been ground, and said inorganic salt is selected from thegroup consisting of metal halides, metal carbonates and metalbicarbonates.
 4. A method as claimed in claim 3, wherein the siliconerubber precursor can be cured or vulcanized at room temperature.
 5. Amethod as claimed in claim 3 or 4, wherein the biologically-acceptablesacrificial filler is bio-compatible, such that it is innately non-toxicand does not leave a toxic residue.
 6. A method as claimed in claim 3 or4, wherein the sacrificial filler does not interact chemically with thesilicone rubber precursor or with the resultant silicone rubber and isstable at temperatures used to cure the resultant mixture.
 7. A methodas claimed in claim 3 or 4, wherein the sacrificial filler is granular.8. A method as claimed in claim 3 or 4, wherein the sacrificial filleris amorphous.
 9. A method as claimed in claim 3, wherein the sacrificialfiller is milled to a particle size of 0.01-10 μm.
 10. A method asclaimed in claim 3, wherein the sacrificial filler is milled in anorganic solvent.
 11. A method as claimed in claim 3, wherein theinorganic salt is selected from the group consisting of lithiumbicarbonate, sodium bicarbonate, potassium bicarbonate, lithiumchloride, sodium chloride and potassium chloride.
 12. A method asclaimed in claim 11, wherein the sacrificial filler is sodiumbicarbonate or sodium chloride.
 13. A method as claimed in claim 12,wherein the sodium bicarbonate or sodium chloride is wet-milled underxylene.
 14. A method as claimed in claim 3, wherein the sacrificialfiller is removed by dissolution.
 15. A method as claimed in claim 3,wherein the sacrificial filler does not cause swelling of the siliconerubber when removed using an aqueous solvent.
 16. A method as claimed inclaim 15, wherein the sacrificial filler is sodium bicarbonate.
 17. Amethod as claimed in claim 3, wherein free —OH groups of the siliconerubber are chemically modified, so as to enhance cell adherence.
 18. Amethod as claimed in claim 3, wherein the surface of the silicone rubberis charged by bombardment with electrons.
 19. A method as claimed inclaim 3, wherein the silicone rubber precursor comprises at least oneadditive that is not removed with the sacrificial filler and serves toimpart desired physical properties to the rubber.
 20. A method asclaimed in claim 19, wherein the additive is a metal powder or carbonblack and serves to render the silicone rubber electrically conductive.21. A method as claimed in claim 20, wherein the additive is stainlesssteel powder.
 22. A method as claimed in claim 20, wherein the additiveis iron oxide.
 23. A method as claimed in claim 19, wherein the additiveis an inert substance, and serves to render the silicone rubbermechanically rigid.
 24. A method as claimed in claim 3, wherein asurface of the silicone rubber precursor is contacted with thesacrificial filler, so as to form a structured silicone rubber having atextured surface.
 25. A method as claimed in claim 24, wherein thetextured surface of the silicone rubber facilitates attachment ofadherent cells.
 26. A method as claimed in claim 24 or 25, wherein thetextured surface of the silicone rubber provides an increased number ofsites for attachment of cells relative to an untextured surface.
 27. Amethod as claimed in claim 3, wherein pores of the silicone rubberprovide sites of attachment for cells.
 28. A method as claimed in claim3, wherein the resultant mixture is shaped prior to curing.
 29. A methodas claimed in claim 3, wherein pores of the silicone rubber are 1 μm-0.5mm in diameter.
 30. A method as claimed in claim 3, wherein the poroussilicone rubber is cut to a desired size or shape.
 31. A method asclaimed in claim 5 wherein the sacrificial filler is crystalline.
 32. Amethod as claimed in claim 3 wherein the sacrificial filler isclassified prior to contacting the silicone rubber precursor.
 33. Amethod as claimed in claim 9, wherein the sacrificial filler is milledto a particle size of 0.05-1 μm.
 34. A method as claimed in claim 9,wherein the sacrificial filler is milled to a particle size of 0.1-0.4μm.
 35. A method as claimed in claim 3, wherein the sacrificial filleris removed by dissolution in an aqueous solvent.
 36. A method as claimedin claim 23, wherein the additive is glass, and serves to render thesilicone rubber mechanically rigid.
 37. A method as claimed in claim 28,wherein the resultant mixture is shaped prior to curing, by molding orextrusion.
 38. A method as claimed in claim 29, wherein the pores are 10μm-0.2 mm in diameter.
 39. A method as claimed in claim 29, wherein thepores are 50 to 150 μm in diameter.